Naturally arising CD4+CD25+ T regulatory cells (Treg) can restrict or alter most types of immune responses (Sakaguchi, 2004, Annu. Rev. Immunol., 22: 531-562). Initially, Treg cells were described to be critical for the control of autoimmunity (Sakaguchi, et al., 1995, J. Immunol., 155: 1151-1164; Shevach, 2000, Annu. Rev. Immunol., 18: 423-449), and were found on adoptive transfer to prevent experimental autoimmune diseases. More recently, Treg have been shown to suppress allogeneic immune responses, and can prevent transplant rejection (Hall, et al., 1998, J. Immunol., 161: 5147-5156; Wood, et al., 2003, Nat. Rev. Immunol., 3: 199-210). In addition, these cells can restrain anti-tumor (Peng L, et al., 2002, J. Immunol., 169: 4811-4821; Gallimore, et al., 2002, Immunology, 107: 5-9), and anti-microbial immune responses (Belkaid Y, et al., 2002, Nature, 420: 502-507). Thus, CD4+CD25+ Treg appear to be central control elements of immunoregulation, and understanding their biology is important to efforts aimed at therapeutically manipulating immune responses.
Treg cells are best characterized in mice where they constitute 5-10% of lymph node and spleen CD4+ T-cell populations. They are generated both through central thymic developmental mechanisms in pathogen free mice, and also arise by poorly defined peripheral generation or expansion mechanisms (Apostolou, et al., 2002, Nat. Immunol., 3: 756-763; Shevach et al., 2002, Nat. Rev. Immunol., 2: 389-400). To date, Treg cells have primarily been defined by co-expression of CD4+ and CD25+ antigens on fresh isolation. CD25 as well as other markers of murine Treg, CTLA4 (CD 152) and GITR (Glucocorticoid Induced TNF-like Receptor), are all activation antigens on conventional T cells, and therefore are not specific. FoxP3, a nuclear protein thought to function as a transcriptional repressor, is a newer marker considered to be more specific for Treg cells (Ramsdell, et al., 2003, Curr. Opin. Immunol., 15: 718-24). It was demonstrated that after activation (T cell receptor based, antigen-specific or anti-CD3), Treg cells can non-specifically suppress proliferation of both CD4+ and CD8+ T cells. The mechanism of suppression is unclear, and in vitro, appears to require cell-cell contact. A functional result of suppression is impaired production of IL-2 (Thornton, et al., 1998, J. Exp. Med., 188: 287-296; Shevach, et al., 2001, Immunol. Rev., 182: 58-67). In vivo, the suppression mechanism is more controversial with some studies demonstrating dependence on immunosuppressive cytokines (Asseman, et al., 1999, J. Exp. Med., 190: 995-1004), which are not required for in vitro suppression.
Studies in mouse models of bone marrow transplantation (BMT) have shown that fresh or culture expanded CD4+CD25+ cells can delay or prevent disease (Taylor et al., 2002, Blood, 99: 3493-3499′ Hoffmann, et al., 2002, J. Exp. Med., 196: 389-399; Cohen, et al., 2002, J. Exp. Med., 196: 401-406). Previous studies have demonstrated that Treg polyclonally expanded ex vivo for 10 days with anti-CD3 plus IL-2, can be effective in preventing graft versus host disease (GVHD; Taylor, et al., 2002, Blood, 99: 3493-3499). Ex vivo expansion of Treg cells with irradiated allogeneic APCs plus exogenous IL-2 is also effective at suppressing GVHD (Cohen, et al., 2002, J. Exp. Med., 196: 401-406). In some model systems, Treg cells can prevent GVHD and still allow for graft versus leukemia (GVL) effects (Edinger, et al., 2003, Nat. Med., 9: 1144-1150; Jones, et al., 2003, Biol. Blood Marrow Transplant, 9: 243-56; Trenado, et al., 2003, J. Clin. Invest., 112: 1688-96). In addition, studies in mouse models of autoimmune disease have demonstrated that culture expanded antigen specific (transgenic TCR) CD4+CD25+ cells can prevent or even treat diabetes (Tang, et al., 2004, J. Exp. Med., 199: 1455-1465). Consequently, Treg cells have a role in clinical immunosuppressive therapy in transplantation, provided human Treg cells can be isolated and expanded in culture to generate sufficient numbers for in vivo infusion.
While the murine data are very promising, there still remains a practical problem of isolating pure Treg from human blood. In young mice, CD4+CD25+ cells are moderately abundant and the CD25+ subset is readily apparent. In humans the CD25+ cells are not as discrete of a population, as there exists a large and overlapping population of CD25-dim cells. It is possible that the co-purification of conventional T cells with Treg is the basis for the modest or variable suppressor activity observed in studies of human CD4+CD25+ cells (Baecher-Allan, et al., 2004, Semin. Immunol., 16: 89-98). FACS cell sorting of the highest 1.7% of CD25+ expressors (CD25high cells) has been reported to enable suppressor cell isolation (Baecher-Allan, et al., 2001, J. Immunol., 167: 1245-1253). A stringent magnetic bead based approach was required to isolate populations of adult blood derived Treg cells pure enough for CD4+CD25+ cells to generate potent suppressor cell lines. Even so, strongly suppressive cell lines could only be generated in a subset (approximately one third) of donors, and potency correlated with cell line purity (Godfrey, et. al., 2004, Blood, 104: 453-461). FACS sorting of CD25high cells (top 2.1%) has been reported to enable more consistent suppressor cell line generation from adult blood (Hoffmann, et al., 2004, Blood. 104: 895-903).
Purification of Treg cells from adult blood is possible, but difficult. Previous attempts using magnetic activated cell sorting (MACS) purification to isolate Treg cells from adult blood that are sufficiently pure for consistent suppressor activity have resulted in variability in cell function. This variability is largely due to the presence of CD25-dim memory cells which overlap with Treg cells. Use of a cell sorter has facilitated the isolation of Treg cells (Baecher-Allan, et al., 2001, J. Immunol., 167: 1245-1253), and enabled the generation of suppressor cell lines from adult blood (Hoffmann, et al., 2004, Blood, 104: 895-903). However, even sorted populations of adult blood derived CD25+ cells (top 2.9%) were found in one report to contain a mix of conventional and regulatory T cells on cloning and functional analysis (Levings, et. al., 2002, J. Exp. Med., 196: 1335-1346).
About 20% of of the CD4+CD25+ adult blood cells express CD45RA. This antigen is not expected to be expressed on suppressor cells, as they have been described in several reports to be CD45RO positive (generally mutually exclusive expression, except for transiently during activation of naïve cells). However, the isolation of these cells was much better than the CD45RA− cells for generating suppressor cell lines (12/12 cell lines isolated by this method were found to be potent suppressors). On naïve T cells the CD45RA splice variant is expressed on the T cell surface. Once a T cell differentiates into a memory cell, it usually expresses the CD45RO isoform (U.S. Publication No. 20050196386).
Cord blood has previously been shown to contain CD4+CD25+ cells by fluorescence activated cells sorting (FACS) (Paganelli, et. al., 1994, Cell Immunol., 155: 486-489; Ng, et al., 2001, Blood 98: 2736-2744; Wing, et al., 2002, Immunology 106: 190-199). However, there is minimal data reported on the function of these cells. One report has inferred suppressive function based on LDA frequency analysis (Ng, et al., 2001, Blood 98: 2736-2744). The only report evaluating functional activity of freshly isolated CD4+CD25+ cells, revealed no suppression of antigen specific responses. In addition, there was no increased antigen specific reactivity of CD4+ cells after CD25+ cell depletion. However, modest suppression was noted in anti-CD3 based T-cell co-culture assays, (60% at 1/1 responder/suppressor cell ratio) (Wing, et al., 2003, Eur. J. Immunol., 33: 579-587). Thus, previous studies indicated that most cord blood derived CD25+ cells were not yet mature enough to be suppressive (Wing K, et al., 2003, Eur. J. Immunol. 33: 579-587).
Accordingly, until the present invention, the properties and benefits of Treg cells were recognized, but method to isolate and generate sufficient numbers of potent suppressor cells were unknown. Therefore, a recognized need for methods to isolate and expand Treg cells existed. The present invention meets this need.